| Biocatalysis is continuing to gain momentum and is now becoming a key component in process chemist. The immobilization of biocatalysts plays an important role in their applications. Recently, inspired by adhesive proteins secreted by marine mussels, various biomimetic adhesive materials which have strong adhesive ability under water were developed. In this study, we prepared different kinds of magnetic nanocomposites with catechol group on surface, and use to immobilize biocatalysts.The factors which dicatated the activity and stability of the immobilized biocatalysts were investigated, and the attachment mechanism of catechol was also determined. The detailed work was introduced as following:(1) Immobilization of ω-transaminase J2315via magnetic catecholic chitosanA novel co-transaminase from Burkholderia cenocepacia J2315has been recruited by homology search and the gene was cloned and functionally expressed in E.coli BL21. The specific activity of purified co-transaminase J2315was8.7U/mg. The enzyme had an optimal temperature of40℃, and an optimal pH of9.0, and it shows (S) enantio-selectivity and good activity towards aromatic aldehydes and aromatic amines. The magnetic catecholic chitosan, which carrying adhesive moieties with strong surface affinity, was prepared by simply coating of catecholic chitosan onto iron oxide nanoparticles, and used to immobilize ω-transaminase J2315. Under optimal conditions,87.5%of the available ω-transaminase J2315was immobilized on the composite, yielding an enzyme loading capacity as high as681.7mg/g. Furthermore, the valuation of enzyme activity showed that ω-transaminase J2315immobilized on CCS-IONPs displayed enhanced pH and thermal stability compared to free enzyme.(2) Immobilization of recombinant E.coli M15/BCJ2315via bio-inspired catecholic chitosanThe recombinant E.coli M15/BCJ2315which harbored a mandelonitrilase from Burkholderia cenocepacia J2315was immobilized via catecholic chitosan and functionalized with magnetism by iron oxide nanoparticles. The immobilized cells showed high activity recovery, enhanced stability and good operability in the enantioselective hydrolysis of mandelonitrile to (R)-(-)-mandelic acid. The ethyl acetate-water biphasic system was built and optimized. Under the optimal conditions, as high as1M mandelonitrile could be hydrolyzed within4h with a final yield and ee value of99%and95%, respectively. Moreover, the successive hydrolysis of mandelonitrile was performed by repeated use of the immobilized cells for6batches, giving a final productivity (g·L-1·h-1) and relative production (g-g-1) of40.9and38.9, respectively.(3) Immobilization of Gluconobacter oxydans using bio-adhesive magnetic nanoparticlesG.oxydans was immobilized via a synthetic adhesive biomimetic material inspired by the protein glues of marine mussels. This approach involves simple coating of a cell adherent polydopamine film onto magnetic nanoparticles, followed by conjugation of the polydopamine-coated nanoparticles to G.oxydans which resulted in cell aggregation. After optimization,21.3mg (wet cell weight) G.oxydans per milligram of nanoparticle was immobilized and separated with a magnet. Importantly, the immobilized cell showed high specific activity and good reusability. The membrane proteins on the surface of G.oxydans, which were attached by polydopamine, were separated via2D electrophoresis and analyzed by MALDI-TOF. The3D structure of these polydopamine-bound proteins revealed that these proteins were rich in Lys, Cys and Arg, which have reactive amine or thiol group toward catechol group.(4) Attachment mechanisms of catecholCoordination between catechol and Fe3+has been investigated, the results revealed the catechol-Fe3+ligands might have three coordination states and stoichiometrically dependent on the pH. The addition of Cys to catechol group suggested that the catechol group can react with thiol group preferentially. Furthermore, the reactivity of o-quinone group toward amine group was pH dependent. However, the thiol group can react with o-quinone under both acid and alkaline condition. The binding of catecholic PEG toward lipase was investigated.The results showed that, under neutral condition, the cathecol PEG can specifically couple to Cys residue which was introduced to the surface of enzyme molecule via site-directed mutagenesis. In alkaline condition, however, the catecholic PEG can react with both amine from Arg or Lys residues and thiol from Cys residue. Besides, the catecholic PEG cannot bind to lipase under acid condition.(5) Catechol-based magnetic affinity nanoparticles:preparation and applicationInspired by the adhesive mechanism of catechol group, which can coordinate with metal ion, as well as react with thiol group specifically under neutral condition, two catechol-based magnetic affinity nanoparticles were designed and synthesized via facile dopamine chemistry. The method involves in-situ coating of iron oxide nanoparticles with polydopamine, followed by conjugation of glutathione or Ni2+to the polydopamine film. The result revealed that GSH-PD-IONPs displayed high selectivity toward GST tagged proteins, and Ni2+-PD-MNPs showed exclusively specificity for His tagged proteins. In addition, the co-transaminase BJ110was selectively immobilized onto Ni2+-PD-MNPs without purification, the immobilized enzyme showed improved specific activity due the affinity property of Ni2+-PD-MNPs toward His tagged proteins. Furthermore, the immobilized enzyme exhibited enhanced stability and reusability.In conclusion, we present a novel approach for biocatalysts immobilization and is based on bio-inspired adhesive materials. It offers the potential advantages of low cost, easy separation, low diffusion resistance, and high efficiency. Furthermore, the approach is a convenient platform technique for immobilization of other biomolecules, such as enzymes, DNA and antibody. Our results may pave a new way to apply the catechol based technique for rational enzyme immobilization, protein modification, multi enzyme conjugation, and intelligent hydrogel preparation. |
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